4-nitropyrrole-2-carboxylic acid amide derivatives

ABSTRACT

Novel 4-nitropyrrole-2-carboxylic acid amide derivatives of the general formula I below: IN WHICH R is selected from the group consisting of hydrogen atom and alkyl groups of one to eight carbons, and R1 is selected from the group consisting of amino, amidino, and GROUPS, R2 and R3 being each selected from the group consisting of hydrogen, alkyl of one to eight carbons, phenyl, and substituted phenyl, and which may be same or different, processes for their preparation, pharmaceutical compositions comprising said derivatives, and a method for using said compositions. These novel derivatives exhibit anti-Trichomonas vaginalis action as well as a very unique action of promoting release of virus out of cells.

I United States Patent Yamabe et al.

[451 Oct. 10, 1972 [54'] 4-NITROPYRROLE-2-CARBOXYLIC ACID AMIDEDERIVATIVES [72] Inventors: Shigeru Yamabe, Toyonaka-shi;

Masanao Shimizu, Kobe-shi; Tadatsugu Yamamoto, Nara-shi, all of [21]Appl. No.: 888,427

[30] Foreign Application Priority Data Dec. 29, 1968 Japan ..43/725 Jan.7, 1969 Japan ..44/l434 [52] US. Cl ..260/240 G, 260/3263, 424/274 [51Int. Cl. ..C07d 27/26 [58] Field of Search ..260/240 G, 240 A, 326.3

[56] References Cited FOREIGN PATENTS OR APPLICATIONS M3,093 2/ l 965France ..260/326.3

OTHER PUBLICATIONS Neth. Application 6,405,652, Nov. 23, 1964; Chem.Abstracts 62zl6l96f.

Primary Examiner-Henry R. Jiles Assistant Examiner-G. Thomas ToddAttorney-Sherman and Shalloway [5 7] ABSTRACT Novel4-nitropyrrole-2-carboxylic acid amide derivatives of the generalformula I below:

in which R is selected from the group consisting of hydrogen atom andalkyl groups of one to eight carhens, and R is selected from the groupconsisting of amino, amidino, and

1t, N=C/ M Rs groups, R and R being each selected from the groupconsisting of hydrogen, alkyl of one to eight carbons, phenyl, andsubstituted phenyl, and which may be same or different, processes fortheir preparation, pharmaceutical compositions comprising saidderivatives, and a method for using said compositions. These novelderivatives exhibit anti-Trichomonas vaginalis action as well as a veryunique action of promoting release of virus out of cells.

2 Claims, 2 Drawing Figures PATENTED B I972 SHEET 1 [IF 2 IO 3040 TO I00TiME AFTER INFECTIOMMINUTE) 7 h H m mma W523 PATEIITEDUCT 1. I972 SHEET2 BF 2 TIME AFTER INFECTION (MINUTE) wwmmm 4-NITROPYRROLE-Z-CARBOXYLICACID AMIDE DERIVATIVES in which R is selected from the group consistingof hydrogen atom and alkyl groups of one to eight carbons, and R isselected from the group consisting of amino, amidino, and

groups, R and R being each selected from the group consisting ofhydrogen, alkyl of one to eight carbons, phenyl, and substituted phenyl,and which may be same or different, are provided.

ln the above general formula I, R is preferably hydrogen atom or analkyl of one to five carbon atoms which may be of straight chain orbranched, e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec.-butyl, tert.-butyl, pentyl or isopentyl group, inter alia., hydrogen ormethyl being preferred. R is selected from the group consisting ofamino, amidino, and

. R2 N=C/ groups in which R and R may be some or different. Preferred Rand R are each selected from the group consisting of hydrogen; straightchain or branched alkyl groups of one to five carbon atoms, e.g.,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert.-butyl, pentyl and isopentyl; phenyl; and substituted phenyl;preferred substituent in phenyl being selected from the group consistingof hydroxyl, carboxyl, straight chain or branched alkyl groups of one tofive carbons, e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec.- butyl, tert.-butyl, pentyl, and isopentyl groups, straight chainor branched alkoxy groups of one to five carbons, e.g., methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, sec.-butoxy, pentyloxy andisopentyloxy groups, halogen atoms, e.g., fluorine, chlorine, bromineand iodine, and nitro group. lt is particularly preferred that R, shouldbe selected from amino, amidino, N- isopropylideneamino,N-(l-phenyl)ethylideneamino, and N-(ortho-hydroxy)benzylideneaminogroups.

Some of the specific 4-nitropyrrole-Z-carboxylic acid amide derivativesmay be named as follows:

4-Nitropyrrole-Z-carboxylic acid hydrazide (compound A).

l-Methyl-4-nitropyrrole-Z-carboxylic acid hydrazide (compound B).2-Amidinocarbamoyl-4-nitropyrrole (compound C). Z-Amidinocarbamoyll-methyl-4-nitropyrrole (compound D). 4-Nitropyrrole-Z-carboxylic acidlidenehydrazide (compound E). l-Methyl-4-nitropyrrole-2-carboxylic acidisopropylidenehydrazide (compound F). 4-Nitropyrrole-2-carboxylic acid-1 -phenylethylidenehydrazide (compound G).l-Methyl-4-nitropyrrole-2-carboxylicacid(orthohydroxy)-benzylidenehydrazide (compound H).

Among the foregoing, particularly preferred compounds for the purpose ofthis invention are B, E, F, and H.

Hereinafter, alkyl groups of one to five carbons may be of straightchain or branched, and include methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec.- butyl, tert.-butyl, pentyl and isopentyl groups.

Also alkoxy groups of one to five carbons may be of straight chain orbranched, and include methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec.- butoxy, pentyloxy, and isopentyloxy groups, etc.

Halogen atoms hereinafter appearing in this specification includechlorine, bromine, iodine, and fluorine.

Such 4-nitropyrrole-2-carboxylic acid amide derivatives of the generalformula I,

isopropyin which R and R possess the same significations as defined inthe foregoing can be prepared by a. reacting the compounds of thegeneral formula II,

in which R has the same signification as defined before, and R isselected from the group consisting of halogen atoms and alkoxy groups ofone to five carbon atoms with the compounds of general formula IIIbelow,

| ii R R in which R, R and R have the same significations as definedbefore in which R has the same signification as defined above, with thecompounds of general formula IV,

in which R and have the, saiiie sigriificationsi defined above.

0. Furthermore, the compounds of general formula Ia, which are withinthe scope of the foregoing.

compounds'of formula I and ia,

l R3 H in which R and R have the same significations as defined before,with the compounds of general formula V,

R,-Y v in which R, has the same signification as defined before, and

Y is selected from the group consisting of halogen atom, sulfuric acidalkyl ester residue and toluenesulfonic acid ester residue.

The reaction of above process a) is advantageously performed, whenacompound of general formula II with halogen as the R, preferablychlorine or bromine, is used as one of the staring compounds, in ananhydrous organicsolvent which is inert under the reaction conditions,e.g., anhydrous benzene, toluene, chloroform, and. the like, preferablyanhydrous benzene, at temperatures ranging from 0 C. to roomtemperature, for several hours, preferably l 3 hours. Anhydrousreactants are favorably employed. It is also permissible to start thereaction under cooling, if so desired, and thereafter continuing thereaction at room temperature. Whereas, when a starting compound ofgeneral formula ll with an alkoxy group of one to five carbon atoms asthe R, preferably methoxy, ethoxy, or propoxy, is used, the reaction canbe performed in a solvent which is inert under the reaction condition,such as water, alcohol, e.g., lower alkanol such as methanol, ethanol,etc., or in the absence of solvent,

under heating to 200 C., preferably C., for 3 10 hours, preferably 6 7hours. It is a preferred practice to perform: the reaction at elevatedpressures, for example, in a sealed tube. The reaction of process (a)can be'effected by using an excessive amount of the compound of generalformula III as an acid binder.

Both the compounds of general formulas la and la produced by theprocesses (b) and (c), and these of general formulas lb and la" used asthe starting materials therein, are included within the compounds ofgeneral formula I produced by the above-described process (a).Therefore, the processes (b) and (c) can be regarded as theinterchanging methods of the compounds of general formula I.

The process (b) can be effected by maintaining the reactants, in thepresence of a solvent which is inert under the reaction conditions, suchas water or alcohol, e.g., lower alkanol such as methanol or ethanol, attemperatures ranging from room temperature to 80 C., preferably 50 80C., for l to 30 minutes, preferably from 1 to 10 minutes. If so desired,the solvent may be replaced by excessive amount of the startingcompound.

The reaction of above process (c) can'be performed by the stepscomprising dissolving the compound of general formula la in-an anhydrousalcohol, for example, lower alkanol such as methanol, ethanol, etc.,containing an alkali metal alkoxide, for example, sodiumorpotassium-methoxide, or sodiumor potassiumethoxide, to convert thecompound of general formula la to the corresponding alkali metal salt,adding further thereto the compound of general formula V, andmaintaining the system at temperatures ranging from room temperature to80 C., preferably at room temperature, for several hours, preferably 4 5hours.

Thus produced compounds of general formulas I, la, and la can berecovered by conventionally employed means, for example,recrystallization.

In the process (a), the compounds of general formulas II and Ill used asthe starting materials can be prepared from known compounds or suchcompounds which can be prepared in the similar manner to the preparationof known compounds.

The starting compounds of general formula lb to be employed in theprocess (b) are those obtained by the process (a), which can be preparedby reacting the compounds of general formula I] with hydrazine inaccordance with said process (a). The compounds of general formula IVare themselves known and readily available.

The compounds of general formula Ia used as the starting materialofprocess (0) are obtained through the process (a) or (b) above. That is,they can be prepared by either reacting the compounds of generalformulalla,

l H-C-R' in which R is selected from the group consisting of halogenatoms and alkoxy groups of one to five carbons with the compounds ofgeneral formula VI,

formula lb H u-CNHNH2 with the compounds of general formula IV inaccordance with the process (b).

The compounds of formula I of this invention can be obtained as freebase, or as addition salt of suitable acid. Such acid addition saltsinclude those of inorganic acids such as sulfates, hydrohalides,perchlorates, nitrates and phosphates; and those of organic acids suchas methanesulfonates, ethanesulfonates, hydroxyethanesulfonates,ethylenesulfonates, benzenesulfonates, toluenesulfonates, andnaphthalenesulfonates, among these sulfates and hydrohalides beingparticularly preferred. These compounds may be applied to the practicaluse inthe form of an acid addition salt as well as in the free baseform.

The novel compounds of general formula I provided in accordance with thepresent invention exhibit interesting chemotherapeutic characteristics,such as demonstrated by ,the tests performed in the conventional manneragainst Trichomonas vaginalis. For example, one of the tests wasperformed as follows: sterile horse serum was added to a culture mediumcomposed of 20 g of yeast extract, 10 g of glucose, 5 g of NaCl, 2 g ofcysteine, 20 g of peptone, 2 ml of methylene blue solution (1 mg/ml),and 1,000 ml of distilled water, and to which diluted liquid of the testcompound was added. Thereafter Trichomonas vaginalis was inoculatedthereto, followed by 4 day cultivation at 37 C. The minimumconcentration of the test compound required for inhibiting the growth ofTrichmonas vaginalis (MIG-Minimum Inhibitory Concentration) was as givenin the table below. At the indicated concentrations, no growth ofTrichomonas vaginalis was observed in the specified culture medium onthe third day from the inoculation.

MIC Against Trichomonas Vaginalis (in vitro) Tested Compound MIC(meg/ml) B l0 D l0 E 30 F G 10 H 10 as Trypanosoma (T. gambiense, T.rhodesiense, T. cru-' 2i), Leishmania (L. donovani, L. tropica, L.braziliensis), Trichomonas (T. buccalis, T. hominis, T. pulmonalia, T.vaginalis), Sporozoa (Plasmodium spp.),

Endamoeba (E. histolytica), etc., particularly as antitrichomonal agentsagainst Trichomonas.

Accordingly, this invention also provides pharmaceutical compositionscontaining as the active ingredient the compounds of general formula Ias mixed with pharmaceutically acceptable organic or inorganic, solid orliquid carriers.

The compositions of the invention can be orally, or parenterally, orlocally administered into the rectum, to men, domestic animals such ascow, pig, and dog, and domestic fowls.

Suitable carriers useful for the compositions include those adjuvantscommonly employed, such as water, lactose, starch, talc, magnesiumstearate, vegetable oil, benzyl alcohol, tragacanth gum, polyalkyleneglycol, etc.

As the compositions to be orally administered, tablets, capsules, pills,granular compositions, syrup and the like prepared in the conventionalmanner can be named. The indicated dose per day of the pharmaceuticallyactive compound in that case ranges from toSOO mg, preferably 300 450mg, which can be administered in one to three divided times per day.

As the compositions to be applied locally or in the rectum, thosecontaining the pharmaceutically effective amount of active ingredientsas mixed with conventionally employed ointment base, such as hydrouslanolin, Vaseline, soft Vaseline, polyethylene glycol, and mixturesthereof, may be used.

It is now found that the compounds of general form ula 1 of theinvention possess the action to promote release of virus out of cells.

Phage, for example, Phage MS2 (Davis J. E., Strauss J. H., andSinsheimer R. L., Science 134, 1427. (1961)), GA and 0,8 (I. Watanabe,T. Miyake, T. Sakurai, T. Shiba and T. Ohno: Proc. Japan Acad. 43, 204(1961)), EAC (T. Nishihara and l. Watanabe; Virus 17, 118 (1967)), and Tare the virus which infect bacteria and propagate in the bacteria. Agarculture medium was solidified as the nutrient source of bacteria, and onwhich soft agar culture medium mixed with bacteria e.g., approximately 2X l0/ml) and phage (approximately 300/ml) was laminated and solidified,followed by an overnight incubation at 37 C. The bacteria grew in theculture medium, causing the latter to become opaque, and at the spotswhere phage propagated, cell lysis took place, forming small circulartransparent spots. Such circles are referred to as plaques.

The number of phage can be measured by counting the number of suchplaques. For example, when 1 ml of liquid containing phage at unknownconcentration is mixed with bacteria in accordance with abovedescribedmethod and the number of plaques formed on the culture medium iscounted, if it is 300, the phage concentration is determined to be 300plaque-forming units/ml (p.f.u). Depending on such factors as type ofthe bacteria to be infected with phage it is not always the case thatone phage particle equals one plaqueforming unit. Therefore, theconcentration of phage is expressed by p.f.u.

When bacteriophage infects the host bacteria, nucleic acid alone of thephage enters into the bacteria, and progeny phage is produced in thebacteria, which causes cell lysis, subsequently causing release of theprogeny phage from the bacteria. The one cycle from infection with phageto the release of phage is referred to one-step growth. Repetition ofone-step growth is referred to multi-step growth. Plaque is anagglomerateof many lysed cells formed by multi-step growth of phage.Therefore, the more the .steps of growth, the greater becomes thediameter of plaque.

We discovered that, when bacteria and phage are mixed in the presence ofthe novel compound of general formula I of the invention and incubatedfor an overnight in culture medium, the diameter of the plaque formedbecomes more than twice greater than the case wherein the novel compoundis absent. This fact clearly proves that, either the novel compound ofthe invention is effectivefor (A) increasing the amount of phagereleased from each host bacterium, or (B) shortening the time requiredfor one-step'growth, i.e., increasing the number of growth cycleperformable within a definite time.

Heretofore, no compound which exhibits the above effect (A) is'known.Whereas, a mutant of T phage, T r, which has shorter time of growthcycle than that of ordinary phage is known [8. Benzer: Proc. Nati.Acad., Sci. U.S.A., 41, 344 (1955)]. However, the growthcycle-increasing effect. is achieved by mutation in genetic property ofphage, and compound having above-described action is yet unknown.

The suitable amount of use of the subject compound for this purposeranges 3O 300 meg/ml, preferably approximately 100 mcg/ml; that ofbacteria ranges l l0 cells/ml, preferably 10 cells/ml; and that ofphage, 100 1,000 p.f.u./ml, preferably 200 p.f.u./ml. The culture mediummay be selected from those conventionally used, which are suited forgrowth of employed bacteria], preferably agar culture medium. Incubationcan be performed in accepted manner, for example, performed at 37C. foran overnight, with satisfactory result.

Furthermore, we carried out the following one-step growth experiment[Ellis, E. L., and M. Delbrueck, J. Gen. Physical, 22, 365 (1939)], inorder to decide whether the plaque-enlargement effect of the subjectnovel compounds is due to the phenomenon (A) or (B) above. Bacteria andphage were mixed in a liquid culture medium into which the novelcompound of this invention had been added. Separately, identicalbacteria and phage were mixed in the same culture medium except that thesubject compound was replaced by distilled water, as a control. Afterincubation at 37 C for minutes the resulting liquid mixtures eachcontained uninfecting phage, phage-infected bacteria andphage-uninfected bacteria. Then phage anti-serum was added to inactiveuninfecting phage,-and immediately thereafter the mixtures were dilutedto at least times to diluted the anti-serum. Such diluting is effectiveto make the antiserum inert to the later-released progeny phage, and theprogeny phage are made uninfecting to the bacteria due to the resultinglow concentration. Thus, 'the phage performs one-step growth only.

The initial stage of infection of bacteria with phage is adsorption ofphage onto the bacterial surface. The probability of this adsorption isdirectly proportional to the product of phage concentration andbacterial concentrationyFor example, if phage and bacteria both at theconcentrations of approximately l-O /ml are mixed as normally practiced,most of the phage adsorb on the bacterial surface within severalminutes. Whereas, if the two are mixed at both the concentrations of 10ml, the adsorption probability is reduced to l/l0 QAccordingly, thediluting to 10 times in the above onestep growth experiment can besafely regarded as reducing the probability of phage-infection ofbacteria to substantially nil.

Phage antiserum bonds with coat protein of phage particles to inactivatephage. Once the nucleic acid of phage enters into bacteria, theantiserum is no more effective. That is, antiserum does not act onphage-infected bacteria, although it is active with uninfecting phageparticles.

The foregoing experiment was further continued as follows. Eachdefinitequantity of the diluted culture media obtained by the described one-stepgrowth was taken at S-minutes or lO-minutes intervals, starting from 10minutes after the infection, and ending after minutes from theinfection. Thus, 15 19 samples taken from the diluted culture medium inwhich the compound of this invention was present, and also 15 19 samplestaken from the diluted culture medium in which the novel compound wasreplaced by distilled water, were obtained. While any suitable number ofsamples may be prepared, 15 19 samples well meet the purpose of theexperiment. Each sample was further diluted with the culture medium to10 10 times, whereby the compound of this invention being inactivated.Subsequently the diluted samples were each mixed with bacteria in softagar culture medium, treated as described in the foregoing, andresulting plaques were counted to determine the respective phageconcentration. In the graph of attached FIG. 1, plaque-formingunits/m]ofeach sample was plotted against the time passage from the infection.From the graph, it can be understood that in case of phage MS2, therelease of progeny phage from the bacteria starts approximately 35minutes after the infection in the control (in which distilled water waspresent instead of the compound of this invention) as indicated by themark while the release-starting time of the progeny phage is advanced by5 10 minutes in the presence of the compound of this invention (AA andthat little appreciable difference exists in average burst size betweenthese curves. This fact demonstrates that the novel compounds of thisinvention possess the foregoing effect B, that is, contribute .toincrease the phage growth cycle within the definite time. Also asaforesaid, if the plaques are counted within approximately 20 minutesafter the infection, phage-infected bacteria can be selectively counted,because no progeny phage is yet released. Whereas, when the plaques arecounted afterapproximately 35 minutes from the infection, the progenyphage released from the infected bacteria can be selectively counted.The average burst size of phage referred in the above means p.f.u. ofthe progeny phage/p.f.u. of the phageinfected bacteria, i.e., averageproduction amount of progeny phage per one infected bacterium. Foreffecting one-step growth of phage, the suitable amount of the novelcompound to be added ranges 30 300 meg/ml, preferably approximately 100meg/ml; bacteria concentration ranges 1O 10 cells/ml. preferably 10cells/ml; and phage concentration is optional, preferably 10 p.f.u./ml.The amount of phage antiserum depends on the antiserum value, which mustbe sufficient for inactivating uninfecting phage. Infection of bacteriawith phage can be sufficiently performed within minutes. At theabove-specified bacterial and phage concentrations, the infection willbe very quickly completed. The subsequent antiserum treatment may begiven for approximately 3 minutes, during 5th 8th minutes after theinfection, with satisfactory result, while only one minute treatment canalso achieve the inactivation of uninfecting phage.

The amount of each sample is variable depending on such factors asconcentration of progeny phage, which is not critical, but preferably0.1 ml.

As above-explained, the novel compounds of this invention exhibit theaction of increasing the number of growth cycle of virus within thedefinite time. In order to determine whether the action is due to B)-l;acceleration of phage-producing rate inside the bacteria, or B)-2;shortening of phage-releasing time from the bacteria, with unchangedphage-producing rate inside the bacteria, the following experiment wasconducted.

As already mentioned, the infection procedure was the same as theone-step growth experiment. Samples of 0.l ml each were taken at varioustime intervals until 60 minutes, starting 10 minutes after theinfection, with satisfactory result. Each sample was added to aconventional mixture for lysing bacteria, for example, a mixture oflysozyme-EDTA and buffer, to cause the lysozymc to act on the bacteriafor 5 30 minutes, preferably 10 minutes, to cause the lysis of bacteria.The lysed mixtures were diluted appropriately. The compound of theinvention employed was inactivated by the dilution mentioned as to theforegoing one-step growth experiment.

The resulting diluted samples were each mixed with bacteria in soft agarculture medium, and treated similarly to the foregoing. The phageconcentration in each sample was measured by counting the plaques. Inthe graph given as FIG. 4, thus determined p.f.u./ml. (phageconcentration) of each sample is plotted against time passage from theinfection. FIG. 4 demonstrates that the amount of phage production isunchanged between the system containing the compound of this invention(AA) and control (Q Whereby the conclusion is drawn that the phagegrowth cycle-increasing effect of the compounds of thisinvention asdemonstrated in FIG. 1 is caused by the foregoing phenomenon B)-2.

Referring to FIG. 2, the area below the dotted line, i.e., the intervalbetween 10 l8 minutes, corresponds to so-called eclipse period.

The above conclusion is drawn through the following procedures. Forexample, under the assumptions that a minutes are required from phageinfection to progeny phage production, and that b minutes are requiredfor the produced progeny phage to be released from the bacteria, FIG. 1indicates that a b is shortened in the presence of the compound of thisinvention. It is apparent that the total number of phage particleswithin and outside the bacteria was counted in the experiment relatingto FIG. 2. Therefore, the p.f.u./ml value resulting from the experimentdepends solely on a, i.e., phage-producing time. Whereas, FIG. 2demonstrates that the phage-producing rate is identical between the Viiare effective. More particularly, the effective derivatives are thosecompounds of general formula, I, inter alia, the compounds specificallynamed in the earlier part of this specification;

Preferred phage include MS2, GA, and OB, and the preferred bacteria isEscherichia coli.

The above activity of the subject compounds of increasing the phagegrowth cycle indicates the following utility of the compounds: reagentfor facilitating plaque formation of virus which difficulty formsplaque, or reagent for enlargement of plaque size for easierobservation. Thus the compounds will make significant contribution inthe field of virology. Furthermore, the compounds can be effectivelyused in the preparation of vaccine against the diseases caused by virus,which requires production of virus in large quantities, for shorteningthe time required for virus growth, and consequently, the time requiredfor the vaccine preparation.

The following examples are given solely for the illustrative purposeofthis invention, but never to restrict the scope thereof.

EXAMPLE 1 4-Nitropyrrole-2-carboxylic acid hydrazide Ethyl4-nitropyrrole-2-carboxylate (1.84 g) was dissolved in 5-10 ml ofethanol, and 6.2 g of hydrazine hydrate was added thereto. In a sealedtube they were reacted at l50 C. for 6-7 hours. The reaction mixture wasconcentrated under reduced pressure to dryness. The residue wassufficiently washed with dilute acetic acid, washed with water anddried. Recrystallization from methanol gave 1.1 g of the intendedproduct in the form of faintly yellowish crystals melting at 263- 264C.

EXAMPLE 2 4-Nitropyrrole-2-carboxylic acid hydrazide a. Preparation of4-nitropyrrole-2-carboxylic acid chloride: 4-Nitropyrrole-2-carboxylicacid (1.5 g) was refluxed in an oil bath for 30-45 minutes together withl 1.9 g of thionyl chloride, and then the excessive thionyl chloride wasdistilled off to give 1.8 g of the intended product.

b. Preparation of 4-nitropyrrole-2-carboxylic acid hydrazide:4-Nitropyrrole-2-carboxylic acid chloride (1.75 g) was dissolved inanhydrous benzene, and the resulting solution was added dropwise to anemulsion of 1.0 g of anhydrous hydrazine in anhydrous benzene at roomtemperature. Precipitated crystals were recovered by filtration andwashed with dilute acetic acid, followed by water-washing and drying.Recrystallization from methanol gave 1.5 g of the intended product inthe form of faintly yellowish crystals melting at 263264 C.

EXAMPLE 3 1-Methyl 4-nitropyrrole-2-carboxylic acid hydrazide Ethyl1-methyl-4-nitropyrrole-2-carboxylate (1.9 g)

was dissolved in 5-10 ml of ethanol, and 6.2 g of hydrazine hydrate wasadded thereto. In a sealed tube they were reacted at 150 C. for 6-7hours.,After completion of the reaction, the reaction mixture wasconcentrated to dryness under reduced pressure. The residue was washedwith water, dried and recrystallized from methanol to give 1.2 g of theintended product in the form of colorless crystals melting at 228'229'C.

EXAMPLE 4 1-Methyl4-nitropyrrole-2-carboxylic acid hydrazide a.Preparation of 1-methyl-4-nitropyrrole-carboxylic acid chloride:1-Methyl-4-nitropyrrole-2-carboxylic acid (1.64 g) was refluxed with11.9 g of thionyl chloride in an oil bath for 30-45 minutes and theexcessive 'thionyl chloride was distilled off to obtain 1.9 g of theintended product.

b. Preparation of l-methyl-4-nitropyrrole-2-carboxylic. acid hydrazide:1-Methyl-4-nitropyrrole-carboxylic acid chloride (1.89 g) was dissolvedin anhydrous benzene, and the solution was added dropwise to an emulsionof 1.0 g of anhydrous hydrazine in anhydrous benzene at room temperaturewith stirring. Precipitated crystals were recovered by filtration,washed with water,'dried and recrystallized from methanol to give 2.0 gof the intended product in the form of colorless crystals melting at228-229 C.

EXAMPLE 5 2-Amidinocarbamoyl-4-nitropyrrole 4-Nitropyrrole-2-carboxylicacid chloride (1.75 g) was dissolved in anhydrous benzene, and thesolution was added dropwise to an emulsion of 1.2 g of guanidineinanhydrous benzene at room temperature with stirring. Precipitatedcrystals were recovered by filtration, washed sufficiently with water,dried and. recrystallized from methanol to give 1.6 g of the intendedproduct in the form of faintly yellowish crystals melting above 270 C.

EXAMPLE 6 Z-Amidinocarbamoyl-1-methyl-4-nitropyrrole1-Methyl-4-nitropyrrole-2-carboxylic acid chloride 1.89 g) was dissolvedin anhydrous benzene, and the solution was added dropwise to an emulsionof 1.2 g of guanidine in anhydrous benzene at room temperature withstirring. Precipitated crystals were recovered by filtration, washedwith water, dried and recrystallized from methanol to give 1.7 g of theintended product in the form of faintly yellowish crystals melting at2l922 1 C.

' 2 EXAMPLE7 4-Nitropyrrole-2-carboxylic lidenehydrazide4-Nitropyrrole-2-carboxylic acid chloride (1.75 g) was dissolved inanhydrous benzene, and the solution was added dropwise to 1.44 g ofacetone hydrazone in anhydrous benzene at room temperature withstirring. Precipitated crystals were recovered by filtration, washedwith water, dried and recrystallized from acetone to give 1.8 g oftheintended product in the form of faintly yellowish crystals melting at247248 C.

acid isopropy- EXAMPLE 8 EXAMPLE 9 4-Nitropyrrole-2-carb0xyliclidenehydrazide 4-Nitropyrrole-2-carboxylic acid hydrazide (1.7 g)prepared in accordance with the method describedin Example 1 or 2 wasrefluxed in acetone for several minutes and filtered. The filtrate wasconcentrated to dryness, and the residue was recrystallized from acetoneto give 1.9 g of the intended product in the form of faintly yellowishcrystals melting at 247248 acid isopropyacid isopropy- EXAMPLE l01-Methyl-4-nitropyrrole-2-carboxy1ic acid isopropylidenehydrazide1-Methy1-4-nitropyrrole-Z-carboxylic acid chloride (1.89 g) wasdissolved in anhydrous benzene, and the solution was added dropwise toacetonehydrazone in anhydrous benzene at room temperature with stirring.Precipitated crystals were recovered by filtration, washed with water,dried and recrystallized from acetone to give 1.8 g of the intendedproduct in the form of faintly yellowish crystals melting at 210-212 C.

EXAMPLE 1 1 1-Methyl-4-nitropyrrole-2-carboxylic acidisopropylidenehydrazide 1-Methy1-4-nitropyrrole-2-carboxylic acidhydrazide (1.8 g) prepared in accordance with the method described inExample 3 or 4 was heated in acetone to dissolve the hydrazide therein,followed by filtration. The filtrate was concentrated to dryness and theresidue was recrystallized from acetone to give 2.1 g of the intendedproduct in the form of faintly yellowish crystals melting at 210-2l2 C.

1.44 g of EXAMPLE 12 l-Methyl-4-nitropyrrole-2-carboxylic acidisopropylidenehydrazide 4-Nitropyrrole-2-carboxylic acidisopropylidenehydrazide (2.1 g) prepared in accordance with the methoddescribed in Example 7, 8 or 9 was suspended in anhydrous ethanolcontaining 0.54 g of sodium methylate. To the suspension, 4.3 gof methyliodide was added dropwise at room temperature, and the mixture wasstirred for 4-5 hours. After completion of the reaction, the reactionmixture was concentrated under reduced pressure, and 5 percenthydrochloric acid was added to the residue. Insoluble matters wereremoved by filtration, washed with water, and dried. The residue waschromatographed over silica gel and the column was eluted with a 1:2mixed solvent of acetone and chloroform. The first eluate was collectedand the solvent was distilled therefrom. Then, the residue wasrecrystallized from acetone to give 0.51 g of the intended product inthe form of faintly yellowish crystals melting at 2 l 2 1 2 C.

EXAMPLE 13 4-Nitropyrrole-2-carboxylic acid(l-phenyl)ethylidenehydrazide 4-Nitropyrrole-2-carboxylic acid hydrazide(1.7 g) prepared in accordance with the method described in Example 1 or2, and 1.32 g of acetophenone were refluxed in methanol. After thesystem had been cooled, precipitated crystals were collected byfiltration and recrystallized from methanol to give 2.5 g of theintended product in the form of faintly yellowish crystals melting at247-249 C.

EXAMPLE 14 l-Methy1-4-nitropyrrole-2-carboxylic acid (orthohydroxy)benzylidenehydrazide l-Methyl-4-nitropyrrole-2-carboxy1ic acid hydrazide(1.84 g) prepared in accordance with the method described in Example 3or 4, and 1.34 g of salicylaldehyde were refluxed in methanol todissolve the hydrazide therein. After the system had been cooled,precipitated crystals were recovered by filtration and recrystallizedfrom methanol to give 2.8 g of the intended product in the form offaintly yellowish needles melting above 260 C.

EXAMPLE l Enlargement effect in the plaque size of phage MS 2 0.1 ml ofan overnight broth culture of E. coli E102 obtained in a customarymanner and 0.1 ml of a phage preparation 1,000-3,000 p.f.u./ml) obtainedby propagation and dilution in a customary manner were mixed with 2.5 mlof a melted soft agar medium containing the compound B, E, F or H (100,ug/ml) (said medium having been prepared from 5 g ofNaCl, 5.5 g ofagar, 20 g of peptone, 2 g of glucose and 1,000 ml of distilled water,and the pH thereof having been adjusted to 7.2 with 1N NaOH aqueoussolution). The resulting mixture was poured onto a petri dish containinga basic agar layer containing the compound B, E, F or H at the sameconcentration as in the soft agar medium (said layer having beenprepared from 15 g of agar,

TABLE 1 Sample compound Enlargement of plaque size Compound B (PY 10)Compound E Compound F Compound H more than twice as great as the normalplaque diameter (of control).

EXAMPLE 16 Enlargement effect in plaque sizes of various phagesExperiments were performed in the same manner as in Example 15 by usingthe compound B and as phage MS2, GA and Q8, respectively. The sizes ofthe resulting plaques were observed. The results are shown in Table 2below.

TABLE 2 Phage Enlargement of plaque size Msz GA QB more than twice asgreat as the normal plaque diameter (control EXAMPLE .17

Effect of compound B on the one-step growth of phage MS2 E. coli B 102grown in PGYC medium (consisting of mixture of 20 g of peptone, 5 g ofNaCl, 2 g of glucose, 0.25 percent of yeast extract 0.01 mole of CaCland 1,000 ml of distilled water, with the pH adjusted to 7.2 with 1NNaOH aqueous solution) at a density of 2.8 X 10 cells/ml was dividedinto 2 aliquots (4.5 ml each). To one of them compound B was added suchthat the concentration of compound B was mcg/ml and the whole volume was5 ml. To the other aliquot was added the same volume of distilled waterinstead of compound B as a control. Both aliquots were infected withphage MS2 at a multiplicity of infection of 1.3 (Multiplicity ofinfection is the ratio of phage concentration: E. coli concentration).Five minutes after the infection, phage MS2 antiserum (K. value: 5,500)was added to each aliquot to form a 100 times dilution of saidantiserum. Phage M52 was subjected to an action of said antiserum for 3minutes, whereby the uninfected phage MS2 was inactivated. Immediatelythereafter, 0.1 ml of each aliquot was diluted 10 -10 times. Sampleswere prepared by taking 0.1 ml of each dilution 10, 12, 15,

MS2 contained in each sample were determined by .counting the number ofresulting plaques. The curves shown in FIG. 1 were obtained by plottingthese values.

The curve drawn by the line AA- is that of the aliquot in which thecompound B was present, and the curve drawn by the line is that of thealiquot in which distilled water was replaced instead of the compound B(control).

EXAMPLE 18 Effect of compound B on the production of intracellular phagePlaques were formed in the same manner as in Example 17 except thatsamples were taken from each aliquot 10, 12.5, 15, 17.5, 20, 22.5, 25,27.5, 30, 32.5, 40 and 60 minutes after the infection, respectively, and0.9 ml ofa lysis mixture [0.7 ml of l/20 M tris-HCl buffer (pH=8.0), 0.1ml of a lysozyme solution (2 mg/ml), 0.1 ml of EDTA (40 mg/ml) and twodrops of chloroform] was added to 0.1 ml of each sample just after theSampling, followed by cell-lysis at 37 C. for minutes. The p.f.u./mlvalues of phage MSZ contained in each sample were determined by countingthe number of the formed plaques. Curves shown in FIG. 2 were obtainedby plotting these values. The curve drawn by the line -4:f A; is that ofthe aliquot in which the compound B was present, and the curve drawn bythe line is that of the aliquot in which distilled water was replacedinstead of the compound B (control).

EXAMPLE l9 Tablets, each containing the following components,

were prepared by the conventional tablet-making method:

l-Methyl-4-nitropyrrole-2- carboxylic acid hydrazide 200 mg Avicel(microcrystalline cellulose) 50 mg Corn starch 46 mg Colloidal silicicacid with hydrolyzed starch 2 mg Magnesium stearate 2 mg Total: 300 mgWe claim: 1. A 4-Nitropyrrole-2-carboxylic acid amide derivatives of theformula wherein R is selected from the group consisting of hydrogen andalkyl of one to eight carbon atoms, and R is R and R eachbeingindependently selected from the group consisting of hydrogen, alkylof one to eight carbon atoms, phenyl, and substituted phenyl wherein thesubstituent is selected from hydroxyl, carboxy], alkyl of one to fivecarbon atoms, alkoxy of one to five carbon atoms, halogen, and nitro.

2. A 4-nitropyrrole-Z-carboxylic acid amide derivatives of claim 1,wherein R is selected from the group consisting of hydrogen and methyl,and R is selected from the group consisting of N-isopropylideneamino,N-(l-phenyl)ethylideneamino, and N-(ortho-hydroxy)benzylideneamino.

2. A 4-nitropyrrole-2-carboxylic acid amide derivatives of claim 1,wherein R is selected from the group consisting of hydrogen and methyl,and R1 is selected from the group consisting of N-isopropylideneamino,N-(1-phenyl)ethylideneamino, and N-(ortho-hydroxy)benzylideneamino.